Non-viral gene delivery has been widely investigated over the past decade as a means to guide tissue regeneration, treat disease and study gene function. However, low efficiencies of gene transfer have limited the use of this approach. In this proposal, we are interested in studying the role that the extracellular matrix environment plays on the ability of mesenchymal stem cells, endothelial cells and smooth muscle cells to become transfected. The interaction of cellular receptors with the various components present in the ECM results in a myriad of cellular responses such as proliferation, migration, differentiation, and apoptosis. We hypothesize that the ECM environment is a key modulator of non-viral gene transfer through inducing cell states that make the cells more or less sensitive to gene transfer and that the role of the ECM will be different for cells seeded in two- versus three dimensions. Our approach is divided into two aims. In aim 1 we will engineer cellular microenvironments (eECMs) that contain multiple components of the ECM to study the effect of the eECMs on non-viral gene transfer. The eECMs will be made using self-assembled monolayers on gold in order to be able to control the amount and conformation of the immobilized proteins and DNA particles. Aside from using reporter plasmids and internalization assays to quantify the efficiency of gene transfer and particle internalization, we will investigate if cellular contractility is involved in the observed changes in gene transfer for different eECMs. In aim 2 we will investigate gene transfer when cells are plated inside a matrix (3D) as opposed to in a flat surface (2D). We believe that understanding how the process of gene transfer differs when the cells are plated inside a matrix from when they are plated in a flat surface is critical for the understanding how to enhance gene transfer in vivo and inside tissue engineered constructs. We will use polystyrene nanoparticles and DNA nanoparticles to study the endocytosis rate and overall gene transfer efficiency in 3D and 2D. We will also investigate if the cellular contractile machinery also plays a role when cells are plated in three dimensions. PUBLIC HEALTH RELEVANCE: Non-viral gene delivery offers is an ideal approach to alter cellular behavior at the molecular level through the delivery of plasmid DNA that encodes for a gene of interest. However, the efficiency of gene transfer is too low to achieve the levels required for therapeutic application. In this proposal we are exploring new avenues to enhance the process of gene transfer through exploiting the natural response of cells to the extracellular matrix.